The ability to deliver nucleic acids carried by delivery vehicles, e.g., recombinant viruses (adenovirus, adeno-associated virus, herpesvirus, retrovirus) which are used with nucleic acid molecules, such as a plasmid, comprising a transgene, to transfect a target cell; molecular conjugate vectors; and modified viral vectors are important for the potential treatment of genetic diseases through gene delivery.
Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb. See generally, Horwitz, M. S., “Adenoviridae and Their Replication,” in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990. Recombinant (adenovirus dodecahedron and recombinant adenovirus conglomerates) to specific cell types is useful for various applications in oncology, developmental biology and gene therapy. Adenoviruses have advantages for use as expression systems for nucleic acid molecules coding for, inter alia, proteins, ribozymes, RNAs, antisense RNA that are foreign to the adenovirus carrier (i.e. a transgene), including tropism for  both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts. See Berkner, K. L., 1992, Curr. Top. Micro Immunol, 158:39-66; Jolly D., 1994, Cancer Gene Therapy, 1:51-64.
Adenoviruses have a natural tropism for respiratory tract cells, which has made them attractive vectors for use in delivery of genes to respiratory tract cells. For example, adenovirus vectors have been and are being designed for use in the treatment of certain diseases, such as cystic fibrosis (CF): the most common autosomal recessive disease in Caucasians. In CF, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disturb cAMP-regulated chloride channel function, resulting in pulmonary dysfunction. The gene mutations have been found to encode altered CFTR proteins which cannot be translocated to the cell membrane for proper functioning. The CFTR gene has been introduced into adenovirus vectors to treat CF in several animal models and human patients. Particularly, studies have shown that adenovirus vectors are fully capable of delivering CFTR to airway epithelia of CF patients, as well as airway epithelia of cotton rats and primates. See e.g., Zabner et al., 1994, Nature Genetics, 6:75-83; Rich et al., 1993, Human Gene Therapy, 4:461-476; Zabner et al., 1993, Cell, 75:207-216; Zabner et al., 1994, Nature Genetics 6:75-83; Crystal et al., 1004, Nature Genetics, 8:42-51; Rich et al., 1993, Human Gene Therapy, 4:461-476.
However, it would be useful to alter the genome of adenovirus, to allow it to be used to deliver a nucleic acid molecule that would be enhanced for expression in the liver, particularly in hepatocytes.
It would be useful to mediate expression of the transgene carried by the adenoviral vector through the use of one or more specialized regulatory elements. In this way the expression of transgene within desired cells can be enhanced and the adenovirus effects can be targeted to certain cells or tissues within an organism.
Like adenoviruses, retroviruses have also been used for delivery of transgenes to target cells. As set forth above, a transgene is a nucleic acid molecule that codes for, inter alia, a protein, RNA, ribozyme, antisense RNA not produced by the virus. Retrovirus virions range in diameter from 80 to 130 nm and are made up of a protein capsid that is lipid encapsulated. The viral genome is encased within the capsid along with the proteins integrase and reverse transcriptase. The retrovirus genome consists of two RNA strands. After the virus enters the cells, the reverse transcriptase synthesizes viral DNA using the viral RNA as its template. The cellular machinery then synthesizes the complementary DNA which is then circularized and inserted into the host genome. Following insertion, the viral RNA genome is transcribed and viral replication is completed.
Examples of retroviruses include Moloney murine leukemia virus (Mo-MuLV), HTLV and HIV retroviruses. Mo-MuLV vectors are most commonly used and are produced simply by replacing viral genes required for replication with the desired transgenes to be transferred. The genome in retroviral vectors contains a long terminal repeat sequence (LTR) at each end with the desired transgene or transgenes in between. The most commonly used system for generating retroviral vectors consists of two parts, the retroviral vector and the packaging cell line.
Retroviruses are typically classified by their host range. For example, ecotropic viruses are viruses which bind receptors unique to mice and are only able to replicate within the murine species. Xenotropic viruses bind receptors found on all cells in most species except those of mice. Polytropic and amphotropic viruses bind different receptors found in both murine and nonmurine species. The host range is determined primarily by the binding interaction between viral envelope glycoproteins and specific proteins on the host cell surface that act as viral receptors. For example, in murine cells, an amino acid transporter serves as the receptor for the envelope glycoprotein gp70 of ecotropic Moloney murine leukemia virus (Mo-MuLV). The receptor for the amphotropic MoMuLV has recently been cloned and shows homology to a phosphate transporter. There are six known receptors for retroviruses: CD4 (for HIV); CAT (for MLV-E (ecotropic Murine leukemic virus E); RAM1/GLVR2 (for murine leukemic virus-A (MLV-A)); GLVRI (for Gibbon Ape leukemia virus (GALV) and Feline leukemia virus B (FeLV-B). RAM1 and GLVR1 receptors are broadly expressed in human tissues.
Retrovirus packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector, i.e., the gag, pol and env genes. For the MMLV vectors, it is the packaging cell line that determines whether the vector is ecotropic, xenotropic or amphotropic. The choice of the packaging cell line determines the cells that will be targeted. Thus, the usefulness of retroviruses for gene transfer is limited by the fact that they are receptor specific.
However, retroviruses are useful for gene delivery systems because they have a high infection efficiency and the retroviral nucleic acid (after reverse transcription) integrates into the host genome resulting in sustained expression of the transgenes carried by the vector. However, typical retroviral vectors are limited in that they require dividing cells for infectivity. Furthermore, in vivo delivery of these vectors is poor and is effective only when infecting helper cell lines. Thus, it would be useful to have a system for increasing the efficiency of retroviral infection.
Certain situations exist where it would be useful to modify the expression of transgenes carried by viruses. For example, tissue-specific expression of the transgene in targeted cells might increase the efficiency of infection, and consequently, a lower volume of virus may be effective in the body. It would be useful to have a method of up-regulating the expression of the transgene in a tissue-specific manner.
One method for targeting specific cell populations to express a protein of interest is to use heterologous regulatory elements that are specifically expressed in the desired target tissue or cell populations. This may be achieved through the use of combinations of tissue-specific enhancers, promoters and/or other regulatory elements. The regulatory elements may be constitutive or inducible, such that they are regulated by the absence or presence of other DNA sequences, proteins or other elements or environmental factors. Where the transgene of interest is a cytotoxic gene, leaky expression would be highly undesirable.